The color and odor of amylases, i.e. of enzymes which catalyze the hydrolysis of starch, can adversely affect the quality of the therapeutic compositions and of the detergent formulations in which they are incorporated.
It is thus desirable that amylase preparations for detergent applications, pharmaceutical applications and food applications be free from components which can cause undesirable color and odor, as well as undesirable haze, instability and allergic activity in the final product. These components may be derived from the microorganisms themselves or from residual fermentation raw materials. In preparations of Gram positive Bacilli, cell wall anionic polymers, peptidoglycans, galactosyl polymers, and other polysaccharide contaminants become solubilized during cell growth due to cell wall turnover. The presence of these bacterial cell wall polymers in amylase preparations can cause several undesirable effects including an increase in the allergenicity, a decrease in amylase stability by binding cations, e.g., Ca.sup.++, and may cause haze formation in detergent formulations. Therefore, it is essential to purify amylases recovered from fermentation broth.
U.S. Pat. No. 5,041,377 discloses a method of producing crystalline subtilisin, i.e. proteases, derived from Bacillus subtilis and Bacillus amyloliquefaciens by the addition of a halide salt to an alkaline subtilisin solution at temperatures less than 10.degree. C., whereby halide salts comprise chlorides, bromides, iodides and fluorides of sodium, potassium or calcium and preferably sodium and calcium chloride.
PCT application WO 91/09943 discloses a method for crystallization of enzymes, such as an amylase, by means of a crystallization agent which is an easily soluble salt of the non-halide type, such as Na, K, Ca, or Mg formate, acetate or nitrate, is added to the aqueous enzyme.
U.S. Pat. No. 4,659,667 discloses a process for producing crystalline alpha-amylase enzyme by concentrating to supersaturation the alpha-amylase solution at a pH in a range near the isoelectric point of the enzyme. However supersaturated solutions are unstable and have to be treated with great care avoiding shocks, dust particles or scratches on the inner wall of the solution's containers. Moreover once the crystallization has been induced by stirring, incubation times of around 72 hours are necessary in order to have approximately 85%-86% of the amylase being in crystallized form.